Differential expression of Type I interferon and inflammatory genes in SARS‐CoV‐2‐infected patients treated with monoclonal antibodies

Abstract Introduction Considering the reported efficacy of monoclonal antibodies (mAbs) directed against the Spike (S) protein of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) in reducing disease severity, the aim of this study was to investigate the innate immune response before and after mAbs treatment in 72 vaccinated and 31 unvaccinated SARS‐CoV‐2 patients. Methods The mRNA levels of IFN‐I, IFN‐related genes and cytokines were evaluated using RT/real‐time quantitative PCR. Results Vaccinated patients showed increased rate of negative SARS‐CoV‐2 PCR tests on nasopharyngeal swab compared with unvaccinated ones after mAbs treatment (p = .002). Unvaccinated patients had lower IFN‐α/ω and higher IFN‐related genes (IFNAR1, IFNAR2, IRF9, ISG15, ISG56 and IFI27) and cytokines (IL‐6, IL‐10 and TGF‐β) mRNA levels compared to vaccinated individuals before mAbs (p < .05 for all genes). Increased IFN‐α/ω, IFNAR1, IFNAR2 and IRF9 levels were observed in unvaccinated patients after mAbs treatment, while the mRNA expression ISGs and IL‐10 were reduced in all patients. Conclusion These data suggest that anti‐S vaccinated patients have increased levels of innate immune genes compared to unvaccinated ones. Also, gene expression changes in IFN genes after mAbs administration are different according to the vaccination status of patients.

Currently available vaccines and therapeutic approaches have proven useful in reducing coronavirus disease 2019 (COVID-19)-associated morbidity and mortality, and to moderate the impact of pandemic on healthcare resources. 1As a cornerstone resource, vaccine provides a stimulus for both humoral and cellular immune responses, required to clear infection and to maintain an immunological memory, 2 but also monoclonal antibodies (mAbs) exhibit a great importance among the best available therapies.Distinct mAbs combinations, including casirivimab/imdevimab and bamlanivimab/etesevimab, received an Emergency Use Authorization from the US Food and Drug Administration for treatment of highrisk outpatients recently diagnosed with mild-tomoderate COVID-19, to reduce viral burden and prevent disease progression. 3,4mAbs against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are designed to bind the receptor-binding domain of Spike (S) protein, preventing the interaction with its receptor angiotensin converting enzyme 2 and entry into the host cell, and promoting its clearance by opsonization. 5lassically, virally infected cells produce Type I interferons (IFN-I), which are involved in the early innate immune response. 6IFN-I bind to their receptor (IFN-α and -β receptor subunit 1 [IFNAR1] and 2 [IFNAR2]), in an autocrine and paracrine manner, and stimulate the phosphorylation and activation of the signal transducer and activator of transcription 1 (STAT1) and 2 (STAT2).When combined with the IFN regulatory factor 9 (IRF9), phosphorylated STAT1 and STAT2 form the IFN-stimulated gene factor 3 complex, which migrates to the nucleous to promote the transcription of hundreds of interferon-stimulated genes (ISGs).ISGs, in turn, inhibit virus multiplication at distinct levels, potentiate the innate immunity, and stimulate an adaptive response. 7,8Several ISGs, such as ISG15, could be induced within the infected cell during acute virus infection even exploiting other ways independent from IFN signaling. 9As a consequence, distinct SARS-CoV-2 proteins are able to cause dysregulation on the IFN-I production and IFN-related genes, allowing virus to escape from such host defenses. 81][12][13] Numerous cytokines and chemokines induced by SARS-CoV-2 infection have been shown to be elevated after vaccination against SARS-CoV-2, although important differences with natural infection need to be considered.Indeed, upon vaccination the inflammatory cytokine response is early and transient, whereas during natural SARS-CoV-2 infection systemic cytokines levels remain elevated throughout COVID-19 clinical course. 14Despite the well-described efficacy and safety of mAbs therapy in SARS-CoV-2infected patients, 15 the effect of this treatment on the IFN-I pathway and inflammatory response is not yet known, nor whether vaccinated and unvaccinated individuals might display a different immunological response to mAbs.
Hence, the aim of this study was to evaluate whether differences exist in the virological response as well as in the levels of IFN-I, IFN-related genes, and cytokines genes between vaccinated and unvaccinated SARS-CoV-2-infected patients after mAbs treatment.In particular, gene expression levels of IFN-I (IFN-α and IFN-ω), IFN-I receptor subunits (IFNAR1 and IFNAR2), IRF9, ISGs (ISG15, ISG56, IFN-α-inducible protein 27 [IFI27]), and cytokines (IL-1β, IL-6, IL-10, TNF-α, and TGF-β) were examined in peripheral blood mononuclear cells (PBMCs) collected from SARS-CoV-2-infected patients before and after mAbs treatment.Moreover, data on gene expression were evaluated according with the vaccination status and production of anti-S antibodies.

| Participants
A longitudinal study at the Division of Infectious Diseases, Department of Public Health and Infectious Diseases, Umberto I Hospital of Sapienza University of Rome (Italy), including 116 patients with polymerase chain reaction (PCR) test confirmed SARS-CoV-2 infection on nasopharyngeal swab from April the 21 and December 10, 2021, discharged to home care, was conducted.All patients received exclusively therapeutic regimens based on combined mAbs, casirivimab (1200 mg) plus imdevimab (1200 mg), or bamlanivimab (700 mg) plus etesevimab (1400 mg), in a single administration, following italian drug agency statement.The data source for patient information analysis was derived from electronic medical records in the Hospital Electronic Information System.The following variables were considered for the study: age, gender, vaccination status against SARS-CoV-2, and Charlson Comorbidity Index. 16A healthy control group, composed of nine individuals matched by age and gender was also included.The study was approved by the Institutional Review Board (Department of Public Health and Infectious Diseases, Sapienza, University of Rome) and the Ethics Committee (Sapienza, University of Rome), and all study participants signed written informed consent.

| PBMC isolation
Fresh peripheral blood samples (20 ml) were collected by venipuncture in Vacutainer tubes containing EDTA (BD Biosciences), from healthy individuals and SARS-CoV-2infected patients at baseline (at least 48 h from diagnosis) (T0) and almost 12 days after mAbs administration (T1), and were processed by Ficoll-Hypaque density gradient centrifugation (Lympholyte, Cedarlane Labs) to obtain PBMCs.PBMCs were washed twice in phosphatebuffered saline and stored at −80°C as dried pellets for RNA extraction.

| Antibody titer against SARS-CoV-2 TrimericS protein quantification
Type G immunoglobulin (IgG) against SARS-CoV-2 Spike protein were determined in infected patients' serum using a commercial assay (LIAISON® SARS-CoV-2 TrimericS IgG).The assay provides anti-S antibody titers as binding antibody units per ml (BAU/mL) and measures between 4.81 and 2080 BAU/mL.Values < 33.8 BAU/ml were considered negative according to the manufacturer's instructions.Specimens containing high levels of anti-TrimericS IgG above the assay measuring range (>2080 BAU/mL) were automatically diluted with a factor of 1:10 using LIAISON® TrimericS IgG Diluent Accessory.In addition, anti-S antibody titers were arbitrarily considered low between 33.8 and 400 BAU/mL, and high for values >400 BAU/mL.

| Statistical analysis
Patients' data were expressed as median (interquartile range) or number (percentage).Demographic, virological, serological, and clinical patients' characteristics were analyzed using "N-1" χ 2 test.Cross-sectional data between vaccinated and unvaccinated patients were analyzed using the Mann-Whitney U test, whereas Wilcoxon signed-rank test for paired samples was used to evaluate longitudinal data between T0 and T1.Spearman's ρ coefficient was calculated to assess the correlation between gene expression levels and vaccination induced antibody titers.A p < 0.05 was considered statistically significant.Statistical analyses were performed using GraphPad Prism software, version 9.4 (GraphPad Software Inc.).

| Levels of IFN-I, IFN-related genes, and cytokines in SARS-CoV-2-infected patients before mAbs treatment according to the vaccination status
To investigate whether previous SARS-CoV-2 vaccination resulted in changes in the expression levels of IFN-I,  Note: Data were analyzed using "N-1" χ 2 test and p < .05were considered statistically significant.
b 12 days after mAbs therapy.Data available for 59 and 26 vaccinated and unvaccinated patients, respectively.
To date vaccines remain the best weapon to fight a pandemic viral infection, as we observed recently with SARS-CoV-2. 19Therapy with mAbs has been proposed for high-risk SARS-CoV-2-infected individuals to prevent progression to severe COVID-19 and reduce hospitalization. 5In this study, we evaluated the expression levels of IFN-I, IFN-related genes and various cytokines in patients before and after mAbs treatment according to the anti-S vaccination status.First, a considerable number (29%) of SARS-CoV-2-vaccinated patients tested negative to SARS-CoV-2 RT-PCR 12 days after mAbs therapy, whereas all unvaccinated patients remained positive and most of them (58%) had C t values of SARS-CoV-2-RNA < 34.In agreement, most of patients with high SARS-CoV-2-RNA levels (C t values ≥ 34) had undetectable anti-S antibodies, whereas an increased rate of negative SARS-CoV-2-RNA tests was observed in those patients with high amount of anti-S antibodies.As a first step for gene expression analysis, we performed a comparison between SARS-CoV-2 patients and healthy donors.This analysis showed that mRNA levels of some genes were slightly higher in patients than in healthy donors, while other genes had similar levels between the two groups (Supporting Information: Figure 2).These results can be in part explained considering that our patients had a clinical symptomatology from absent to mild, so the production of IFN pathways and cytokines might be not highly dysregulated as reported in patients with severe COVID-19.Interestingly, we found that IFN-α and IFN-ω mRNA levels were reduced in unvaccinated compared to vaccinated individuals before mAbs treatment.These differences might be due to a potential IFN-I protection role observed in vaccinated subjects, as suggested for yellow fever vaccine. 20Indeed, a previous anti-S vaccination might permit a better control of early virus replication allowing IFN-I to act immediately against SARS-CoV-2 and avoiding the viral escape mechanisms developed to overcome innate immunity.By contrast, IFNAR1 and IFNAR2 mRNA levels were increased in unvaccinated individuals compared with vaccinated ones.In our opinion, in vaccinated patients the IFN response appears to be ready, controlled, and driven by the effects of the vaccine, as suggested previously. 21In unvaccinated patients, the expression of different components of IFN pathway seems to be altered; in particular, the host inability to efficiently contrast the early stages of SARS-CoV-2 infection could allow the virus to inhibit IFN-I production 8 and promote the IFN receptors disruption.In agreement, Hayn et al. 22 found that viral protein nsp14 reduce in vitro IFNAR1 expression on surface of SARS-CoV-2-infected cells, favoring its lysosomal degradation.Therefore, the increased gene expression of IFNAR1/2 observed in unvaccinated patients might be an immune defense mechanism of the infected cell to physiologically subvert the reduction in the expression of receptor caused by SARS-CoV-2.Furthermore, an increase in IFNAR1/2 expression might allow that, although IFN-1 levels are reduced, more receptor ligand interactions could form, increasing the production of several ISGs.Notably, the unvaccinated patient's group exhibited higher mRNA levels of IRF9, ISG15, ISG56, and IFI27, despite reduced IFN-I production compared with vaccinated ones.In this context, IFN-independent stimulation of these ISGs, might be due to the SARS-CoV-2 itself or mediated by the action of other cytokines. 9In this complex scenario, unvaccinated patients had increased IL-6, IL-10, and TGF-β mRNA levels before mAbs treatment, suggesting a state of major inflammation during SARS-CoV-2 infection compared with the vaccinated ones.In support of this hypothesis, IL-6 is one of the key mediators of inflammation and is considered a central mediator of toxicity in cytokine release syndrome, 23 a major cause of fatal outcome in COVID-19. 24Despite the well-known anti-inflammatory properties of IL-10, evidence from literature showed a COVID-19 severity prediction role for this cytokine, together with the pro-inflammatory IL-6. 25,26In addition, TGF-β is an anti-inflammatory cytokine produced by most immune cells during SARS-CoV-2 infection, but an adequate immune response is inhibited by its overproduction, as reported in several respiratory viral infections. 27,28o further assess whether the vaccination status might modulate the innate immune response, SARS-CoV-2infected patients were stratified according to anti-S antibody production.We found that anti-S antibody-positive patients exhibited increased IFN-α and IFN-ω mRNA levels than anti-S antibody-negative ones.A positive correlation was found between anti-S antibody titers and IFN-α mRNA production, indicating that anti-S vaccine induce adaptive immunity, but also innate response as previously reported. 29At the same time, anti-S antibody-positive patients had lower IRF9, ISG15, ISG56, IFI27, and IL-10 mRNA levels than anti-S antibody-negative ones, with asserted negative correlation between these genes and antibody titers.Given these findings, as anti-S antibody levels decreased, the expression of almost immune and inflammatory genes analyzed in this study appears to be closer to that of unvaccinated patients.
As far as the analysis of the effects of mAbs on immune response was concerned, the transcript levels of IFN-α and IFN-ω did not change after mAbs treatment in vaccinated patients, whereas unvaccinated group showed increased levels of these genes, which reached levels similar to those of the vaccinated one.Probably the SARS-CoV-2-RNA clearance caused by mAbs therapy in unvaccinated patients could have promoted IFN-I response at levels similar to those of vaccinated patients.In this regard, IFNAR1, IFNAR2, and IRF9 mRNAs were reduced in the unvaccinated group at T1, whereas the mAbs treatment did not result in any relevant changes in the expression of these genes in the vaccinated one.Moreover, mAbs treatment decreased ISG15, ISG56, IFI27, and IL-10 mRNA in both groups.Therefore, these results confirm that mAbs treatment favor the SARS-CoV-2-RNA disappearance and suggest that this therapy might participate indirectly in the modulation of the expression of IFN-I, IFN-related gene, and the inflammation status in unvaccinated patients, whereas the expression of most of the immune genes analyzed, with the exception of ISGs and IL-10 mRNAs, could be attenuated and independent from mAbs treatment in vaccinated patients.Given the enhanced innate immune response observed after vaccination, 30 and these results, we speculated that anti-S vaccinated patients are immunological advantaged compared with unvaccinated patients, who are able to reach the immune status of the former group in term of expression of IFN and cytokines genes only after mAbs administration.Data from literature showed a decrease in IFN-I protein levels in unvaccinated patients 5 days after casirivimab/imdevimab treatment. 31However, compared with our study design, there were differences in mAbs administration timing and follow-up analysis.Moreover, levels of IFN-I were measured as proteins and nor as mRNAs.
From a clinical point of view, a previous study found that mAbs therapy with casirivimab/imdevimab was associated with a low rate of hospitalization in both vaccinated and unvaccinated individuals. 32Although IFN-I mRNA expression did not change in our vaccinated patients after mAbs treatment, a relevant number of patients had undetectable SARS-CoV-2 RNA, maybe due to the combined antiviral effect of previous vaccination and mAbs.By contrast, despite the increased IFN-I mRNA expression levels in the unvaccinated group following mAbs treatment, none were negative for SARS-CoV-2-RNA 12 days after the treatment.A recent study reported that high-risk SARS-CoV-2 patients treated with nirmatrelvir/ritonavir had more rapid conversion from positive to negative SARS-CoV-2 RT-PCR test compared with nontreated patients, without significant differences based on the vaccination status. 33

| CONCLUSIONS
Our findings confirm that mAbs treatment is effective against SARS-CoV-2 infection, reducing viral RNA to low or undetectable levels 12 days after mAbs treatment, mostly in those patients that have received a previous anti-S vaccination.Furthermore, mAbs treatment promotes an increase in the IFN-α/ω levels and IFN-related genes reduction in SARS-CoV-2-infected unvaccinated patients, restoring them to the expression levels found in vaccinated patients before treatment.

| LIMITATION OF THE STUDY
Despite the significant outcomes in the evaluation of innate immunity in mAbs treated SARS-CoV-2 patients, this study has some limitations.SARS-CoV-2 vaccines showed to differ each other in term of safety and efficacy, evaluated in adverse events and neutralizing antibodies production, respectively, 34 but most of our patients were vaccinated with BNT162b2 and the other vaccines percentages were too small to assess eventual differences.We only evaluated IFN, IFN-related genes, and cytokines mRNA expression, whereas it would have been interesting to evaluate them at protein level.The evaluation of mAbs effect on IgG subclasses would also have been very interesting, but, unfortunately, the serological test used does not allow discrimination of IgG subclasses.Moreover, further analysis based on cellular immunity are needed to better define the immunological effects of mAbs treatment in SARS-CoV-2-infected patients.
T A B L E 1 Demographic, virological, serological, and clinical characteristics of SARS-CoV-2-infected patients.